Lateral-flow assay device having flow constrictions

ABSTRACT

A lateral-flow assay device includes a substrate having a sample addition zone and a wash addition zone downstream thereof along a fluid flow path through which a sample flows. The fluid flow path is configured to receive a wash fluid in the wash addition zone. A hydrophilic surface is arranged in the wash addition zone. Flow constriction(s) are spaced apart from the fluid flow path and arranged to define, with the hydrophilic surface, a reservoir configured to retain the wash fluid by formation of a meniscus between the hydrophilic surface and the flow constriction(s). The fluid flow path draws the wash fluid from the reservoir by capillary pressure. Apparatus for analyzing a fluidic sample and methods of displacing a fluidic sample in a fluid flow path of an assay device are also described.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application claims priority under applicable portions of 35 U.S.C.§ 119 to U.S. Patent Application Ser. No. 62/034,825, filed Aug. 8, 2014and entitled: LATERAL-FLOW ASSAY DEVICE HAVING FLOW CONSTRICTIONS, theentire contents of which are herein incorporated by reference.

TECHNICAL FIELD

This application relates to the field of clinical diagnostics and morespecifically to lateral-flow assay devices.

BACKGROUND

The use of diagnostic assays is very well known for the diagnosis,treatment and management of many diseases. In that regard, differenttypes of diagnostic assays have been developed to simplify the detectionof various analytes in clinical samples such as blood, serum, plasma,urine, saliva, tissue biopsies, stool, sputum, skin or throat swabs andtissue samples or processed tissue samples. These assays are frequentlyexpected to provide a fast and reliable result, while being easy to useand inexpensive to manufacture.

One common type of disposable assay device includes a sample additionzone or area for receiving the liquid sample, at least one reagent zone(also known as a conjugate zone), a reaction zone (also known as adetection zone), and optionally an absorbing zone. These zones can bearranged in order along a fluid passage or channel. These assay devices,commonly known as lateral test strips, can employ a porous material,e.g., nitrocellulose, defining a path for fluid capable of supportingcapillary flow. Examples include those devices shown in U.S. Pat. Nos.5,559,041, 5,714,389, 5,120,643, and 6,228,660, all of which areincorporated herein by reference in their entireties.

The sample addition zone of these assay devices frequently includes aporous material, capable of absorbing the liquid sample, and, whenseparation of blood cells is required, also effective to trap the redblood cells. Examples of such materials are polymeric membrane filtersor fibrous materials, such as paper, fleece, or tissue, comprising e.g.,cellulose, wool, glass fiber, asbestos, synthetic fibers, polymers, ormixtures of the same.

Another type of lateral-flow assay device is defined by a non-poroussubstrate having a plurality of upwardly extending microposts (alsoreferred to as “micropillars” or “projections”). The microposts aredefined dimensionally and in terms of their spacing to produce capillaryflow when a liquid is introduced. Examples of such devices are disclosedin U.S. Pat. No. 8,025,854B2, WO 2003/103835, WO 2005/089082, WO2005/118139 and WO 2006/137785, all of which are incorporated byreference herein in their entireties.

A known non-porous assay device of the above type is shown in FIG. 1.The lateral-flow assay device 1 has at least one sample addition zone 2configured to receive a sample 101, graphically represented using ateardrop shape. The sample 101 can include, e.g., a bodily fluid orother fluid to be tested for an analyte. The lateral-flow assay device 1also includes a reagent zone 3, at least one detection zone 4, and atleast one wicking zone 5, each disposed on a common substrate 9. Thezones 2, 3, 4, 5 are aligned along a defined fluid flow path 64 by whichthe sample 101 or a portion thereof flows from the sample addition zone2 to the wicking zone 5 under the influence of capillary pressureprovided between ones of a -plurality of microposts 7. Capture elements,such as antibodies, can be supported in the detection zone 4, theseelements being capable of binding to an analyte of interest, the captureelements being deposited on the device, e.g., by coating. The term“element” is not limited to atoms, i.e., chemical elements of theperiodic table, but can also refer to molecules, e.g., of ionically orcovalently-bonded atoms, or other chemical compounds or biologicalsubstances. In addition, a labeled conjugate material, also capable ofparticipating in reactions that will enable determination of theconcentration of the analyte, is separately deposited on the device inthe reagent zone 3, wherein the conjugate material carries a label fordetection in the detection zone 4 of the lateral-flow assay device 1.

The conjugate material is gradually dissolved as the sample 101 flowsthrough the reagent zone 3, forming a conjugate plume of dissolvedlabeled conjugate material and sample 101 that flows downstream alongthe defined fluid flow path 64 of the lateral-flow assay device 1 to thedetection zone 4. As the conjugate plume flows into the detection zone4, the conjugated material will be captured by the capture elements suchas via a complex of conjugated material and analyte (e.g., as in a“sandwich” assay) or directly (e.g., as in a “competitive” assay).Unbound dissolved conjugate material will be swept past the detectionzone 4 and into the wicking zone 5.

An instrument such as that disclosed in U.S. 2006/0289787A1, U.S.2007/0231883A1, U.S. Pat. Nos. 7,416,700 and 6,139,800, all incorporatedby reference in their entireties herein, is configured to detect thebound conjugated material in the detection zone 4. Common labels includefluorescent dyes that can be detected by instruments which excite thefluorescent dyes and incorporate a detector capable of detecting theresulting fluorescence. In the foregoing devices and in the conductionof assays, the resulting level of signal in the detection zone is readusing a suitable detection instrument after the conjugate material hasbeen dissolved and the sample 101 and unbound conjugate material havereached and subsequently filled the wicking zone 5 of the lateral-flowassay device 1.

In a typical point of care (POC) lateral flow assay format, it isdesirable to remove unbound conjugate materials to lower backgroundsignal and improve assay accuracy. In some assays, fluid of the sample101 continues to flow through the detection zone 4 after all thedissolved conjugate passes the detection zone 4. In this way, theflowing sample 101 removes unbound conjugate materials. However,endogenous interferents may be present in the sample 101 that mayinterfere with assay results (e.g., hemoglobin, bilirubin of aparticular patient). For these assays, wash fluid separate from thesample 101 can be applied to remove the interferent from the detectionzone 4 or other parts of the detection channel. Moreover, some assaysinvolve pre-mixing the conjugate material with the sample 101 prior toaddition of the mix to the sample addition zone 2 to obtain a longerincubation time. For these types of assays, since the sample 101 ismixed with the conjugate, a wash fluid is applied to remove unboundconjugate from the detection zone 4. In these and other embodiments,wash fluid can be formatted or designed to provide an acceptable wash.Accordingly, adding wash fluid is necessary for some selected assays in,e.g., a POC lateral flow format.

However, adding wash reagent is a challenge in various priorlateral-flow assay devices. The wash fluid is to flow in the gapsbetween pillars (or in the pores of a porous structure, such ascellulose acetate). However, since flow resistance in gaps or pores ismuch larger than outside of the pillar matrix (or porous) structure,wash fluid cannot be “pushed” into the fluid flow path 64 (the pillarmatrix) to accomplish the wash. Wash fluid has to be “pulled” into thegaps between pillars or pores of a porous material by the capillarypressure. There is therefore a need for assay devices and ways of usingassay devices that are more compatible and usable with various washfluids.

BRIEF DESCRIPTION

According to one aspect, there is provided a lateral-flow assay devicecomprising:

a) a substrate having a sample addition zone and a wash addition zonedisposed along a fluid flow path through which a sample flows undercapillary action in a downstream direction away from the sample additionzone and towards the wash addition zone, wherein the fluid flow path isconfigured to receive a wash fluid in the wash addition zone;

b) at least one hydrophilic surface arranged in the wash addition zone;and

c) one or more flow constriction(s) spaced apart from the fluid flowpath and arranged to define, with the at least one hydrophilic surface,a reservoir configured to retain the wash fluid by formation of ameniscus between the hydrophilic surface and the one or more flowconstriction(s); wherein the fluid flow path is configured to draw thewash fluid from the reservoir by capillary pressure.

According to another aspect, there is provided apparatus for analyzing afluidic sample, the apparatus comprising:

a) at least one assay device including a sample addition zone and a washaddition zone disposed along a fluid flow path;

b) a sample-metering mechanism configured to selectively apply thefluidic sample to the sample addition zone;

c) a wash-metering mechanism configured to selectively apply a washfluid to the wash addition zone, wherein the wash addition zone includesone or more flow constriction(s) spaced apart from the fluid flow pathto form a meniscus in the applied wash fluid;

d) at least one measurement device; and

e) a controller configured to operate each of the sample-meteringmechanism, wash-metering mechanism, and at least one measurement devicein accordance with a predetermined timing protocol in order to determineat least one characteristic of the applied fluidic sample, wherein thecontroller operates the wash-metering mechanism after operating thesample-metering mechanism.

According to still another aspect, there is provided a method ofdisplacing a fluidic sample in a fluid flow path of an assay device, themethod comprising:

dispensing the fluidic sample from a sample supply onto a sampleaddition zone of the assay device, wherein the dispensed fluidic sampletravels along the fluid flow path of the assay device; and

dispensing a wash fluid from a wash-fluid supply onto a wash additionzone of the assay device downstream of the sample addition zone alongthe fluid flow path so that a meniscus is formed in the dispensed washfluid by at least one flow constriction of the assay device, wherein thefluid-flow path draws dispensed wash fluid out of a reservoir defined atleast partly by the meniscus and the drawn wash fluid displaces at leastsome of the fluidic sample in the fluid-flow path.

Various aspects advantageously provide an effective supply of the washfluid to the fluid flow path, even in the face of variations in the rateof wash-fluid delivery or the volume of wash fluid delivered. Variousaspects advantageously restrict the wash fluid from flowing outside thepillar (or other porous) structures of the fluid flow path. Variousaspects advantageously effectively restrict the flow of the samplethrough the fluid flow path, which can improve the accuracy of assays.

These and other features and advantages of various embodiments,variations, and modifications will be readily apparent from thefollowing Detailed Description, which should be read in conjunction withthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a known lateral-flow assay device;

FIG. 2 is a plan view of another known lateral-flow assay device;

FIG. 3 illustrates a plan view of a lateral-flow assay device made inaccordance with at least one embodiment;

FIG. 4 is a plan view of details of a wash addition zone of alateral-flow assay device according to an exemplary embodiment;

FIG. 5 is a front elevational section along the line V-V in FIG. 4 andshows flow constrictions according to an exemplary embodiment;

FIG. 6 is a side elevational section along the line VI-VI in FIG. 4 andshows wash fluid ingress into a fluid flow path according to anexemplary embodiment;

FIGS. 7 and 8 are plan views of exemplary groove configurations in washaddition areas according to various embodiments;

FIGS. 9 and 10 are elevational sections of an exemplary lateral-flowassay device according to various embodiments and illustrate stages inwhich fluid fills an internal volume of the assay device;

FIG. 11A is a sectioned perspective of a lateral-flow assay deviceaccording to various aspects;

FIG. 11B is an elevational section along the line XIB-XIB in FIG. 11A;

FIG. 12 is an elevational section of an exemplary lateral-flow assaydevice illustrating effects of contact angle;

FIG. 13 is an elevational section of an exemplary lateral-flow assaydevice illustrating stages in which fluid fills an internal volume ofthe lateral-flow assay device;

FIG. 14 is an elevational section of another exemplary lateral-flowassay device;

FIGS. 15-27 are perspectives of components of lateral-flow assay devicesaccording to various aspects;

FIGS. 28-30 are graphical representations of photographs of stages in anexperimental test of an exemplary lateral-flow assay device according tovarious aspects;

FIGS. 31-33 are graphical representations of photographs of stages inanother experimental test of an exemplary lateral-flow assay deviceaccording to various aspects;

FIGS. 34-36 are graphical representations of photographs of stages inyet another experimental test of an exemplary lateral-flow assay deviceaccording to various aspects;

FIG. 37 is a schematic of an apparatus for analyzing a fluidic sampleaccording to at least one exemplary embodiment, and related components;

FIG. 38 shows a flowchart illustrating an exemplary method fordisplacing a fluidic sample in a fluid flow path of an assay device; and

FIG. 39 is a high-level diagram showing components of a data-processingsystem in accordance with various embodiments.

DETAILED DESCRIPTION

The following description relates to certain embodiments for a washaddition area design for a lateral-flow assay device. It will be readilyapparent that the embodiments described herein are intended to be merelyexemplary and therefore numerous other variations and modifications arepossible. In addition, several terms are used throughout the followingdiscussion such as “first”, “second”, “above”, “below”, “top”, “bottom”,“lateral” and the like for purposes of providing a suitable frame ofreference in regard to the accompanying drawings. To that end, theseterms should not be regarded as being overly restrictive in terms of thescope of the described apparatus and methods, unless otherwisespecifically indicated herein.

It should further be noted that the accompanying drawings are notnecessarily presented to scale and therefore no narrowing interpretationshould be made in terms of dimensions that have been depicted.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” are intended to further include pluralreferents unless the context clearly dictates otherwise.

The term “about” as used in connection with a numerical value throughoutthe description and the claims denotes an interval of accuracy, familiarand acceptable to a person skilled in the art. The interval governingthis term is preferably ±30%.

In terms of defining certain of the terms that follow, the term“analyte” is used as a synonym of the term “marker” and intended tominimally encompass any chemical or biological substance that ismeasured quantitatively or qualitatively and can include smallmolecules, proteins, antibodies, DNA, RNA, nucleic acids, viruscomponents or intact viruses, bacteria components or intact bacteria,cellular components or intact cells and complexes and derivativesthereof.

The term “sample” herein means a volume of a liquid, solution orsuspension, intended to be subjected to qualitative or quantitativedetermination of any of its properties, such as the presence or absenceof a component, the concentration of a component, etc. Typical samplesin the context of the present invention as described herein are human oranimal bodily fluids such as blood, plasma, serum, lymph, urine, saliva,semen, amniotic fluid, gastric fluid, phlegm, sputum, mucus, tears,stool, etc. Other types of samples are derived from human or animaltissue samples where the tissue sample has been processed into a liquid,solution, or suspension to reveal particular tissue components forexamination. The embodiments of the present invention are applicable toall bodily samples, but preferably to samples of whole blood, urine orsputum.

In other instances, the sample can be related to food testing,environmental testing, bio-threat or bio-hazard testing, etc. Thisrepresents only a small example of samples that can be used in thepresent invention.

As described herein, determinations based on lateral flow of a sampleand the interaction of components present in the sample with reagentspresent in the device or added to the device during the procedure, anddetection of such interaction, either quantitatively or qualitatively,may be for any purpose, such as diagnostic purposes. Such tests areoften referred to as “lateral flow assays”.

Examples of diagnostic determinations include, but are not limited to,the determination of analytes, also called markers, specific fordifferent disorders, e.g., chronic metabolic disorders, such as bloodglucose, blood ketones, urine glucose (diabetes), blood cholesterol(atherosclerosis, obesity, etc.); markers of other specific diseases.,e.g., acute diseases, such as cardiac coronary infarct markers (e.g.,troponin I, troponin-T, NT-proBNP), markers of thyroid function (e.g.,determination of thyroid stimulating hormone (TSH)), markers of viralinfections (e.g., the use of lateral flow immunoassays for the detectionof specific viral antibodies), etc.

Yet another important field of assays is the field of companiondiagnostics in which a therapeutic agent, such as a drug, isadministered to an individual in need of such a drug. An appropriateassay is then conducted to determine the level of an appropriate markerto determine whether the drug is having its desired effect.Alternatively, assay devices as described herein can be used prior toadministration of a therapeutic agent to determine if the agent willhelp the individual in need.

Yet another important field of assays is that of drug tests, for easyand rapid detection of drugs and drug metabolites indicating drug abuse.Exemplary assays include the determination of specific drugs and drugmetabolites in a urine or other sample.

The term “lateral-flow assay device”, as discussed herein, refers to anydevice that receives fluid, such as at least one sample, such as abodily fluid sample, and includes at least one laterally disposed fluidtransport or flow path along which various stations or sites (zones) areprovided for supporting various reagents, filters and the like throughwhich sample traverses under the influence of capillary or other appliedforces and in which lateral flow assays are conducted for the detectionof at least one analyte of interest.

The terms “automated clinical analyzer”, “clinical diagnostic apparatus”or “clinical analyzer,” as discussed herein, refer to any apparatusenabling the scheduling and processing of various analytical testelements, including lateral-flow assay devices, as discussed herein, andin which a plurality of test elements can be initially loaded forprocessing. Such apparatus can include a plurality of components orsystems configured for loading, incubating and testing/evaluating aplurality of analytical test elements in automated or semi-automatedfashion and in which test elements are automatically dispensed from atleast one contained storage supply, such as a cartridge, without userintervention.

The term “testing apparatus” refers to any device or analytical systemthat enables the support, scheduling and processing of lateral-flowassay devices. A testing apparatus can include an automated clinicalanalyzer or clinical diagnostic apparatus such as a bench, table-top ormain frame clinical analyzer, as well as point of care and othersuitable devices. For purposes of this application, the testingapparatus may include a plurality of components or systems for loading,testing, or evaluating at least one lateral-flow assay device, includingdetection instruments for detecting the presence of at least onedetectable signal of the assay device.

The terms “zone”, “area” and “site” are interchangeably used in thecontext of this description, examples and claims to define parts of afluid flow path on an assay device, either in prior art devices oraccording to an embodiment described herein, including devices in whicha sample is first applied to the device and then subsequently directed.The term “reaction” is used to refer to any interaction that takes placebetween components of a sample and reagent(s) on or in the substrate, orbetween two or more components present in the sample. The term“reaction” is in particular used to define a reaction taking placebetween an analyte and a reagent as part of the qualitative orquantitative determination of the analyte.

The terms “substrate” or “support” refers to the carrier or matrix towhich a sample is added, and on or in which the determination isperformed, or where the reaction between analyte and reagent takesplace.

The term “detection” and “detection signal” refers herein to the abilityto provide a perceivable indicator that can be monitored either visuallyand/or by machine vision such as a detection instrument (e.g., afluorimeter, reflectometer or other suitable device).

Referring to FIG. 2, there is shown one version of a lateral-flow assaydevice 20 including a planar substrate 40 which can be made from amoldable plastic or other suitable non-porous material. Further detailsof this and related devices are described below and in U.S. PatentApplication Publication No. 2014/0141527 A1, entitled “Quality/ProcessControl of a Lateral-flow assay device Based on Flow Monitoring,” whichis incorporated herein by reference in its entirety.

The substrate 40 is defined by a top surface 44, which is furtherdefined by a fluid flow path 64. The fluid flow path 64 includes aplurality of discrete areas or zones in spaced relation to one anotherincluding a sample addition zone 48, a reagent zone 52, a plurality ofdetection zones 56 located in a detection channel 55 (for clarity, onlyone detection zone 56 is shown) and a receiving or wicking zone 60.According to this design, each of the above-noted zones are fluidlyinterconnected with one another in linear fashion along at least onedefined fluid flow path 64 and in which a plurality of microposts 7,FIG. 1, are disposed within at least one of the zones and/or the fluidflow path 64, the microposts 7 extending upwardly from either the lowersurface of the fluid flow path 64 or the discrete zones defined on thelateral-flow assay device 20.

The microposts 7 are preferably dimensioned to induce lateral capillaryflow, wherein the microposts 7 preferably include a height, diameterand/or center to center spacing to induce fluidic flow along the atleast one fluid flow path. In one version thereof, the microposts 7 canbe sufficiently dimensioned so as to induce capillary flow as aso-called “open” structure without the need for additional structure(i.e., side walls, cover or lid) or the application of any externallyapplied forces. According to this specific design, a defined fluid flowpath 64 is created, extending from the sample addition zone 48 to thewicking zone 60. The illustrated fluid flow path 64 extendssubstantially in a straight-line fashion between the sample additionzone 48 and the wicking zone 60. In other configurations, the fluid flowpath 64 can include one or more lateral bends or turns.

As noted and in various embodiments, the defined fluid flow path 64 isat least partially open, or entirely open. As noted above and by “open”what is meant is that there is no lid or cover which is maintained at adistance that would contribute to capillary flow. Thus a lid, if presentas physical protection for the fluid flow path 64 and the lateral-flowassay device 20, is not required to contribute to the capillary flow inthe flow path. According to this specific design, a hydrophilic layer 70can be directly applied to the top of the microposts 7 in the wickingzone 60 in order to increase fluid flow in the lateral-flow assay device20 and in which a plurality of vents 72 can be defined in thehydrophilic layer 70. The hydrophilic layer 70 can include a plasticbacker tape (not shown) and a hydrophilic adhesive (not shown) on theside of the backer tape arranged to face the fluid flow path 64. Invarious examples, a flow promoter 57 is arranged in the fluid flow path64 bridging the edge of the hydrophilic layer 70 to promote flow underthe hydrophilic layer 70 placed over the wicking zone 60.

Various examples of flow promoters, mixers, flow restrictors, and otherstructures useful for controlling flow in the fluid flow path 64 aredescribed in U.S. Patent Application Ser. No. 62/035,083, filed Aug. 8,2014, the disclosure of which is incorporated herein by reference in itsentirety. That application describes examples of size and shapecharacteristics of the sample addition zones 48 according to variousaspects, features in the reagent zone 52 to effect more efficientdissolution according to various aspects, a curved portion of the fluidflow path 64 configured to mix fluid passing through the fluid flow path64 according to various aspects, and features in the wicking zone 60including flow promoters similar to the flow promoter 57 according tovarious aspects.

An open lateral flow path is described including the defined microposts7, for example, in the following published applications: WO 2003/103835,WO 2005/089082; WO 2005/118139; WO 2006/137785; and WO 2007/149042, allof which are incorporated by reference in their entireties. Theextending microposts 7 have a height, diameter and a distance ordistances between the microposts 7 such that lateral capillary flow ofan applied fluid, such as plasma, preferably human plasma, in the zonehaving the microposts 7 is achieved. These relationships are discussedin U.S. Pat. No. 8,821,812, which is incorporated by reference in itsentirety.

In addition to optimizing the above-mentioned height, diameter and adistance or distances, the above-noted microposts 7 may be given adesired chemical, biological or physical functionality, e.g. bymodifying the surface of the microposts 7 for purposes, for example, ofthe reagent zone(s) 52 and detection zone(s) 56 of the lateral-flowassay device 20. In one embodiment, the microposts 7 have a height inthe interval of about 15 to about 150 μm, preferably about 30 to about100 μm, a diameter of about 10 to about 160 μm, preferably 40 to about100 μm, and a gap or gaps between the microposts 7 of about 3 to about200 μm, preferably 5 to 50 μm or 10 to about 50 μm from each other. Thefluid flow path 64 between the sample addition zone 48 and the wickingzone 60 may have a length of about 5 to about 500 mm, preferably about10 to about 100 mm, and a width of about 0.3 to about 10 mm, preferablyabout 0.3 to about 3 mm, preferably about 0.5 to 1.5 mm. The microposts7, according to this device design, are substantially cylindrical interms of their configuration and cross section. However, their specificdesign of the microposts 7 can also easily be varied to those ofdifferent shapes (e.g., rhombic, hexagonal, etc) and sizes to augmentflow, as well as to filter materials.

Still referring to FIG. 2, the sample addition zone 48 can receive afluid sample 101, FIG. 1, from a liquid dispenser, such as a pipette orother suitable device. The sample is typically deposited onto the top ofthe sample addition zone 48. In various embodiments, a filter material(not shown) is placed within the sample addition zone 48 to filterparticulates from the sample or to filter blood cells from blood so thatplasma can travel through the lateral-flow assay device 20. In theseembodiments, the sample is typically deposited onto the filter material.

The sample then flows, e.g., via capillary action of the microposts, tothe reagent zone 52, which can include reagent(s) useful in thereaction, e.g., binding partners such as antibodies or antigens forimmunoassays, substrates for enzyme assays, probes for moleculardiagnostic assays, or auxiliary materials such as materials thatstabilize the integrated reagents, materials that suppress interferingreactions, and the like. Generally, one of the reagents useful in thereaction bears a detectable signal as discussed herein. In some cases,the reagents may react with the analyte directly or through a cascade ofreactions to form a detectable signal such as a colored or fluorescentmolecule. In one preferred embodiment, the reagent zone 52 includesconjugate material. The term “conjugate” means any moiety bearing both adetection element and a binding partner.

For purposes of this description, a detection element is an agent whichis detectable with respect to its physical distribution and/or theintensity of the signal it delivers, such as but not limited toluminescent molecules (e.g., fluorescent agents, phosphorescent agents,chemiluminescent agents, bioluminescent agents and the like), coloredmolecules, molecules producing colors upon reaction, enzymes,radioisotopes, ligands exhibiting specific binding and the like. Thedetection element also referred to as a label is preferably chosen fromchromophores, fluorophores, radioactive labels and enzymes. Suitablelabels are available from commercial suppliers, providing a wide rangeof dyes for the labeling of antibodies, proteins and nucleic acids.There are, for example, fluorophores spanning practically the entirevisible and infrared spectrum. Suitable fluorescent or phosphorescentlabels include for instance, but are not limited to, fluoroceins, Cy3,Cy5 and the like. Suitable chemiluminescent labels include but are notlimited to luminol, cyalume and the like.

Similarly, radioactive labels are commercially available, or detectionelements can be synthesized so that they incorporate a radioactivelabel. Suitable radioactive labels include but are not limited toradioactive iodine and phosphorus; e.g., ¹²⁵I and ³²P.

Suitable enzymatic labels include but are not limited to horseradishperoxidase, beta-galactosidase, luciferase, alkaline phosphatase and thelike. Two labels are “distinguishable” when they can be individuallydetected and preferably quantified simultaneously, without significantlydisturbing, interfering or quenching each other. Two or more labels maybe used, for example, when multiple analytes or markers are beingdetected.

The binding partner is a material that can form a complex that can beused to determine the presence of or an amount of an analyte. Forexample, in a “sandwich” assay, the binding partner in the conjugate canform a complex including the analyte and the conjugate and that complexcan further bind to another binding partner, also called a captureelement, integrated into the detection zone 56. In a competitiveimmunoassay, the analyte will interfere with binding of the bindingpartner in the conjugate to another binding partner, also called acapture element, integrated into the detection zone 56. Example bindingpartners included in conjugates include antibodies, antigens, analyte oranalyte-mimics, protein, etc.

As the sample interacts with the reagent in the reagent zone 52, thedetection material begins to dissolve in which a resultant detectablesignal is contained within the fluid flow, which is subsequently carriedinto the adjacent detection zone 56.

Still referring to FIG. 2, the detection zone 56 is where any detectablesignal can be read. In a preferred embodiment and attached to themicroposts 7 in the detection zone 56 are capture elements. The captureelements can hold binding partners for the conjugate or complexescontaining the conjugate, as described above. For example, if theanalyte is a specific protein, the conjugate may be an antibody thatwill specifically bind that protein to a detection element such asfluorescence probe. The capture element could then be another antibodythat also specifically binds to that protein. In another example, if themarker or analyte is DNA, the capture molecule can be, but is notlimited to, synthetic oligonucleotides, analogues, thereof, or specificantibodies. Other suitable capture elements include antibodies, antibodyfragments, aptamers, and nucleic acid sequences, specific for theanalyte to be detected. A non-limiting example of a suitable captureelement is a molecule that bears avidin functionality that would bind toa conjugate containing a biotin functionality. The detection zone 56 caninclude multiple detection zones. The multiple detection zones can beused for assays that include one or more markers. In the event ofmultiple detection zones, the capture elements can include multiplecapture elements, such as first and second capture elements. Theconjugate can be pre-deposited on the lateral-flow assay device 20, suchas by coating in the reagent zone 52. Similarly, the capture elementscan be pre-deposited on the lateral-flow assay device 20 on thedetection zone 56. Preferably, both the detection and capture elementsare pre-deposited on the lateral-flow assay device 20, or on the reagentzone 52 and the detection zone 56, respectively.

Downstream from the detection zone 56 and along the fluid flow path 64is the wicking zone 60. The wicking zone 60 is an area of thelateral-flow assay device 20 with the capacity of receiving liquidsample and any other material in the flow path, e.g. unbound reagents,wash fluids, etc. The wicking zone 60 provides a capillary pressure tocontinue moving the liquid sample through and out the intermediatedetection zones 56 of the lateral-flow assay device 20. The wicking zone60 and other zones of the herein described lateral-flow assay device 20can include a porous material such as nitrocellulose, or alternativelycan be a non-porous structure defined by the microposts 7, as previouslydescribed. The wicking zone 60 can further include non-capillary fluiddriving means, such as an evaporative heater or a pump. Further detailsof wicking zones as used in lateral-flow assay devices 20 according tothe various embodiments are found in U.S. Pat. No. 8,025,854 and U.S.Patent Application Publication No. 2006/0239859 A1, both of which areincorporated herein by reference in their entireties.

Tests (assays) are typically completed when the last of the conjugatematerial has moved into the wicking zone 60 of the lateral-flow assaydevice 20. At this stage, a detection instrument, such as a fluorimeteror similar device, is used to scan the detection zone 56, the detectioninstrument being, e.g., incorporated within a portable (hand-held orbench top) testing apparatus. The detection instrument that can be usedto perform the various methods and techniques described herein canassume a varied number of forms. For example, a mainframe clinicalanalyzer can be used to retain a plurality of lateral-flow assay devicesas described in copending U.S. Patent Application Publication No.2013/0330713 A1, the entire contents of which are herein incorporated byreference. In a clinical analyzer at least one detection instrument,such as a fluorimeter, can be provided, for example, in relation to anincubator assembly as a monitoring station in which results can betransmitted to a contained processor.

In various examples, the instrument can include a scanning apparatusthat is capable of detecting fluorescence or fluorescent signals.Alternatively, an imaging apparatus and image analysis can also be usedto determine, for example, the presence and position of at least onefluorescent fluid front of a lateral-flow assay device. According to yetanother alternative version, infrared (IR) sensors could also beutilized to track the position of fluid position in the lateral-flowassay device. For instance, an IR sensor could be used to sense the˜1200 nm peak that is typically associated with water in the fluidsample 101 to verify that sample had indeed touched off onto thesubstrate of the lateral-flow assay device. It should be readilyapparent that other suitable approaches and apparatus capable ofperforming these techniques could be utilized herein.

The microposts 7, FIG. 1, are preferably integrally molded into thesubstrate 40 from an optical plastic material such as ZEONOR®, suchthrough an injection molding or embossing process. The width of thedetection channel 55 in the fluid flow path 64 is typically on the orderof about 0.5 mm to about 4 mm, and preferably on the order of about 2mm. Other portions of the fluid flow path 64 according to variousexamples can have widths of less than about 0.5 mm, or on the order ofabout 0.5 mm to about 4 mm, or greater than about 4 mm. Widths of about1 mm can also be used for the detection channel 55, provided sufficientsignal for a suitable detection instrument, such as a fluorimeter, canbe read even if the reagent plume does not cover the entire width of thedetection zone 56.

Components of the lateral-flow assay devices (i.e., a physical structureof the device whether or not a discrete piece from other parts of thedevice) described herein can be prepared from copolymers, blends,laminates, metalized foils, metalized films or metals. Alternatively,device components can be prepared from copolymers, blends, laminates,metalized foils, metalized films or metals deposited one of thefollowing materials: polyolefins, polyesters, styrene containingpolymers, polycarbonate, acrylic polymers, chlorine containing polymers,acetal homopolymers and copolymers, cellulosics and their esters,cellulose nitrate, fluorine containing polymers, polyamides, polyimides,polymethylmethacrylates, sulfur containing polymers, polyurethanes,silicon containing polymers, glass, and ceramic materials.Alternatively, components of the device can be made with a plastic,elastomer, latex, silicon chip, or metal; the elastomer can comprisepolyethylene, polypropylene, polystyrene, polyacrylates, siliconelastomers, or latex. Alternatively, components of the device can beprepared from latex, polystyrene latex or hydrophobic polymers; thehydrophobic polymer can comprise polypropylene, polyethylene, orpolyester. Alternatively, components of the device can comprise TEFLON®,polystyrene, polyacrylate, or polycarbonate. Alternatively, devicecomponents are made from plastics which are capable of being embossed,milled or injection molded or from surfaces of copper, silver and goldfilms upon which may be adsorbed various long chain alkanethiols. Thestructures of plastic which are capable of being milled or injectionmolded can comprise a polystyrene, a polycarbonate, or a polyacrylate.In a particularly preferred embodiment, the lateral-flow assay devicesare injection molded from a cyclic olefin polymer (COP), such as thosesold under the name Zeonor®. Preferred injection molding techniques aredescribed in U.S. Pat. Nos. 6,372,542, 6,733,682, 6,811,736, 6,884,370,and 6,733,682, all of which are incorporated herein by reference intheir entireties.

Still referring to FIG. 2, the defined fluid flow path 64 of thelateral-flow assay device 20 or other lateral-flow assay devicesdescribed herein can include open or closed paths, grooves, andcapillaries. In various embodiments, the fluid flow path 64 comprises alateral flow path of adjacent ones of the microposts 7, FIG. 1, having asize, shape and mutual spacing such that capillary flow is sustainedthrough the flow path. In one embodiment, the flow path is in a channelwithin the substrate 40 having a bottom surface and side walls. In thisembodiment, the microposts 7 protrude from the bottom surface of thefluid flow path 64. The side walls may or may not contribute to thecapillary action of the liquid. If the sidewalls do not contribute tothe capillary action of the liquid, then a gap can be provided betweenthe outermost microposts 7 and the sidewalls to keep the liquidcontained in the flow path defined by the microposts 7. Preferably, thereagent that is used in the reagent zone 52 and the capture members ordetection agent used in the detection zone 56 is bound directly to theexterior surface of the microposts 7 used in the herein describedlateral-flow assay device 20.

FIG. 3 illustrates a plan view of a lateral-flow assay device 300 inaccordance with at least one embodiment. The lateral-flow assay device300 includes the substrate 9 having the sample addition zone 2 and awash addition zone 409. The sample addition zone 2 and the wash additionzone 409 are disposed along the fluid flow path 64, through which thesample 101, FIG. 1, flows under capillary action in a flow direction F(“downstream”) away from the sample addition zone 2 and towards the washaddition zone 409. The fluid flow path 64 is configured to receive awash fluid 301 (represented in phantom) in the wash addition zone 409.For example, the lateral-flow assay device 300 can include a coverhaving an opening for passage of wash fluid, as discussed below. Inanother example, the fluid flow path 64 can be an open-channel flow pathopen to receipt of wash fluid from above.

The lateral-flow assay device 300 includes at least one hydrophilicsurface 308 arranged in the wash addition zone 409. The hydrophilicsurface 308 is useful with an aqueous wash fluid 301. In an example, thesubstrate 9 includes, or is coated with or bonded to, a material withwhich the wash fluid 301 has a contact angle of less than 45°. As usedherein, the term “hydrophilic surface” refers specifically to a surfacethat is wetted by the wash fluid 301. In at least one example, the washfluid 301 includes numerous surfactants that permit the wash fluid 301to wet certain types of plastic that are hydrophobic to pure water.Hydrophilic surfaces such as the hydrophilic surface 308 can includesuch plastics, which are hydrophilic with respect to the wash fluid 301.

The lateral-flow assay device 300 also includes one or more flowconstriction(s) 310. As used herein, a “flow constriction” is astructural feature that assists in containing the wash fluid 301 withinthe wash addition zone 409 or that assists in restricting the wash fluid310 from spreading out of the wash addition zone 409. Some exemplaryflow constrictions narrow the cross-section of flow across thehydrophilic surface 308 or otherwise impede, resist, or arrest (even ifonly temporarily) the flow of the wash fluid 301 across the hydrophilicsurface 308. Examples of flow constrictions include a nozzle nearing thesubstrate 9, and the substrate 9 turning a corner out of plane, e.g., atthe edge of a groove in the substrate 9. Such flow constrictions arediscussed below. The flow constriction(s) 310 are spaced apart laterallyfrom the fluid flow path 64, as shown more clearly in FIG. 4.

The flow constriction(s) 310 are arranged to define, with the at leastone hydrophilic surface 308, a reservoir 535 (FIG. 5) configured toretain the wash fluid 301 by formation of a meniscus between thehydrophilic surface 308 and the one or more flow constriction(s) 310, asdiscussed below. The fluid flow path 64 is configured to draw the washfluid from the reservoir by capillary pressure.

As discussed above with reference to FIGS. 1 and 2, the lateral-flowassay device 300 can include, e.g., in the fluid flow path 64, aplurality of the microposts 7, FIG. 1. The microposts 7 can extendupwardly from the substrate 9 proximal to the wash addition zone 409 orother zones described herein. The microposts 7 have heights, diametersand reciprocal spacing between the microposts 7 that induce lateralcapillary flow of the sample 101, the wash fluid 301, or both. Moreover,the lateral-flow assay device 300 can include at least one reagent zone303, disposed along the fluid flow path 64 downstream of the sampleaddition zone 2.

Furthermore, the lateral-flow assay device 300 can include at least onedetection zone 56 disposed along the fluid flow path downstream of thesample addition zone 2 and the wash addition zone 409. The at least onedetection zone 56 can include a detection material responsive to ananalyte of the sample 101 to produce a detectable signal, as discussedbelow with reference to FIG. 37.

Referring to FIG. 4, there is shown a plan view of an exemplarylateral-flow assay device 400 according to various embodiments. Inaspects such as that shown, groove(s) are used as the only flowconstriction(s) 310. For example, the lateral-flow assay device 300 canbe an open-top lateral-flow assay device, i.e., a lateral-flow assaydevice with no cover.

In this example, the substrate 9 includes the at least one hydrophilicsurface 308. The one or more flow constriction(s) 310 include at leastone groove 410 formed in the hydrophilic surface 308, and laterallywithin the wash addition zone 409. In various examples, the grooves 410can be elongated, straight or curved, short, circular or elliptical, orother shapes (when viewed from above). In at least one example, thegrooves 410 are elongated and have widths between 50 μm and 200 μm. Inother examples, the widths of the grooves 410 can be between 5 μm and1000 μm, or can be greater than 1000 μm.

In the example shown, the lateral-flow assay device 400 includes aplurality of the flow constriction(s) 310, each of the flowconstrictions 310 including groove(s) 410 formed in the hydrophilicsurface 308. The groove(s) 410 are arranged along respective arcuatepaths 411 about the centerline 464 of the fluid flow path 64. Therespective arcuate paths 411 can be circular, elliptical, or anothershape. Circular grooves advantageously provide greater stability, sincecapillary pressure operates to pull the wash fluid 301 into a circularconfiguration in the absence of flow constriction(s) 310. Accordingly,in at least one example, the grooves 410 are circularly arcuate in shapeto maintain a round fluid dome above the fluid flow path 64. Thegeometric center of the arcuate path for each of the grooves 410 ispreferably on the geometric centerline of the fluid flow path 64 if thefluid flow path 64 is straight, as in this example, so that the washfluid 301 enters the fluid flow path 64 symmetrically along thecenterline of the fluid flow path 64.

As noted above with reference to FIG. 3, the grooves 410 are spacedapart laterally from the fluid flow path 64. This spacing advantageouslyrestricts or impedes the wash fluid 301 or the sample 101 in the fluidflow path 64 from flowing into the grooves 410 by capillary pressure.

In various examples, such as that shown, the flow constriction(s) 310,FIG. 3, include at least three spaced-apart grooves 410, e.g., fourspaced-apart grooves, formed in the hydrophilic surface 308. In variousexamples, such as that shown, at least one of the grooves 410 isarranged along a substantially arcuate path 411 disposed substantiallyabout a portion of the fluid flow path 64. Aspects using a plurality ofgrooves advantageously are more robust to different wash fluid volumes(e.g., permitting a reduction in the precision with which volumes of thewash fluid 301 should be metered) or provide increased reliability ofmaintaining the dome shape of the reservoir 535, FIG. 5, in case theinner groove is covered by the wash fluid 301 during dispensing, or dueto imperfections in the grooves 410 that permit the spread of the washfluid 301 over the substrate 9.

FIG. 5 is a front elevational section along the line V-V in FIG. 4 andshows flow constrictions according to various aspects. The substrate 9has the fluid flow path 64 recessed therein. Two of the flowconstrictions 310, FIG. 3, are the grooves 410 recessed into thesubstrate 9 within the area covered by the hydrophilic surface 308. Theillustrated grooves 410 have substantially rectangular cross-sections.The wash fluid 301 wets the hydrophilic surface 308 to form adome-shaped meniscus 520, 530 above the fluid flow path 64 due tocapillary pressure and surface tension. The volume of a reservoir 535bounded by the meniscus 520, 530 is variable, depending on the volume ofthe wash fluid 301 delivered by a wash-metering mechanism 3725, FIG. 37.The sizes of the meniscus 520, 530 and the reservoir 535 shrink as thewash fluid 301 is drawn from the reservoir 535 into the fluid flow path64 to perform the wash.

The reservoir 535 provides a stable meniscus that advantageouslyaccommodates a wide range of volumes of the delivered wash fluid (e.g.,between 7 and 17 μL) while keeping substantially the same washperformance. Another advantage of a fluid meniscus 520, 530 is that itcan buffer large variations in the delivery rate of the wash fluid 301.The grooves 410 in the illustrated embodiment also advantageously assistin maintaining a round shape of the wash fluid 301 in the reservoir 535at the hydrophilic surface 308.

In a hypothetical example using a wash fluid 301 with a contact angle of45° against the hydrophilic surface 308, if the hydrophilic surface 308were flat and did not have the grooves 410 (graphically represented bythe dotted lines across the tops of the grooves 410), a meniscus 520(shown stippled) would form. The contact angle of 45° in thishypothetical example is shown at an angle 521 with respect to thehorizontal hydrophilic surface 308.

In an example using the wash fluid 301 with the contact angle of 45° andwith the grooves 410, a meniscus 530 forms. The 45° contact angle isshown at an angle 531 with respect to the vertical edge of the grooves410. The volume under the meniscus 530 is the reservoir 535 defined bythe groove(s) 410, i.e., the flow constriction(s), and the at least onehydrophilic surface 308. The reservoir 535 is configured to retain thewash fluid 301 by formation of the meniscus 530 between the hydrophilicsurface 308 and the one or more flow constriction(s) 310.

The wash fluid 301 is drawn around the corner 511 by surface tension andcontact forces, and consequently the cross-sectional area of the flow isrestricted. The grooves 410 and the resultant angle 531 raise themeniscus 530 compared to the meniscus 520. This increases the radius ofthe reservoir 535, increasing or substantially increasing the volume ofthe reservoir 535. For example, hemispherical reservoirs have thevolumes indicated in Table 1, below, for various radii. As can be seen,increasing radius rapidly increases volume.

TABLE 1 R (mm) 1 1.25 1.5 1.75 2 V (μL) 2.1 4.1 7.1 11.2 16.7

Accordingly, using the grooves 410 or similar flow constrictions 310surrounding the fluid flow path 64 can advantageously permit maintainingand controlling the shape of the meniscus 530 and of the reservoir 535to increase the volume of the reservoir 535 above the fluid flow path64.

FIG. 6 is a side elevational section along the line VI-VI in FIG. 4, andshows ingress of the wash fluid 301 into the fluid flow path 64. Thereservoir 535 is shown arranged over the fluid flow path 64, and holdingthe wash fluid 301. In this example, the fluid flow path 64 includes themicroposts 7 arranged over the substrate 9. The sample 101 has filled atleast a portion of the fluid flow path 64. In various examples, the washfluid 301 enters fluid flow path 64 from the reservoir 535 proximate theedge of the reservoir 535, e.g., being drawn by capillary pressurebetween the microposts 7.

In various exemplary configurations using the microposts 7, the washfluid 301 is dispensed into the fluid flow path 64 between the sampleaddition zone 2 and the detection zone 56 to interrupt or displace thefluid of the sample 101. The wash fluid 301 forms a dome shaped meniscusabove the fluid flow path 64 so that fresh wash fluid 301 enters thefluid flow path 64 from above the fluid flow path 64 while the flow ofsample stops flowing toward the reaction zone. A dome shaped wash fluidmeniscus above the fluid flow path 64 is advantageous since the flowresistance is the smallest from above the fluid flow path 64 as comparedwith sample fluid flowing through between the microposts 7. This lowflow resistance will stop sample flow while supplying fresh wash fluid301 from the front edge of the reservoir 535. Prior geometry designsusing a shallow well in a wash addition area do not reliably maintainthe dome shape of the dispensed wash fluid 301. The wash fluid 301 caneasily spread and result in a thin layer of the wash fluid 301 above thefluid flow path 64 instead of a dome, especially when the wash fluid 301has a low contact angle for the hydrophilic surface 308, FIG. 3 (e.g. ifthe contact angle is 45°). In this case, wash efficiency is poor sincelittle of the wash fluid 301 above the fluid flow path 64 is availableand sample 101 will continue to flow even after the addition of washfluid. Configurations described herein advantageously maintain thereservoir 535 to effectively supply the wash fluid 301 to the fluid flowpath 64.

Specifically, in at least one example, the wash fluid 301 has a largeamount or a relatively high concentration of surfactants. Thesesurfactants are useful for washing, but increase the difficulty ofdrawing from a thin layer of the wash fluid 301 into the fluid flow path64. Accordingly, in this example it is preferable to maintain a bulkfluid in the reservoir 535 from which the fluid flow path 64 can draw.The grooves 410, FIG. 5, advantageously increase the size of thereservoir 535, permitting more effective flow of the wash fluid 301 intothe fluid flow path 64 than in prior schemes with no flow constrictions.

In the example of FIG. 5, the sample 101 has filled the fluid flow path64, the wash fluid 301 has been applied, and the wash fluid 301 hasbegun to displace the sample 101 in the fluid flow path 64. The washfluid 301 can flow both downstream (along the flow direction F) andupstream (opposite the flow direction F). In various aspects, the washfluid flows faster downstream than upstream. In an example, there is anarea 655 of the fluid flow path 64 at least partly under the reservoir535 in which there is no flow, i.e., in which the contents of the fluidflow path 64 are stagnant.

Referring to FIG. 7, there is shown a plan view of an exemplary grooveconfiguration of a lateral-flow assay device 700 according to variousembodiments. The fluid flow path 64 in the exemplary lateral-flow assaydevice 700 has a 90° bend in the wash addition zone 409. In thisexample, at least one of the grooves 410 is disposed substantially abouta reference point 710 along a centerline 764 of the fluid flow path 64leaving the wash addition zone 409. This placement of the referencepoint 710, i.e., the geometry center of the grooves 410, advantageouslymaintains symmetry in the flow of the wash fluid 301 along the fluidflow path 64 departing the wash addition zone 409.

Also as shown here, it is not required that each of the grooves 410 orother flow constriction(s) 310, FIG. 3, have the same width W or otherdimensions. In this example, the grooves 410 are arranged alongsubstantially arcuate paths (not shown) having respective radii, e.g.,radii R1, R2, R3, with respect to the reference point 710.

Referring to FIG. 8, there is shown a plan view of an exemplary grooveconfiguration of a lateral-flow assay device 800 according to variousembodiments. In this example, at least one of the groove(s) 410 isconfigured as a segment of a spiral. The segment can have any length andnumber of turns (for the avoidance of doubt, fractional turns andgrooves 410 with less than one full turn can be used). As a result, oneor more of the groove(s) 410 can be a spiral passing through more than360° of rotation around a center point. However, this is not required.

In the example shown, the grooves 810 are arranged along a spiral path869. The spiral path 869 is arranged to laterally extend on either sideof the fluid flow path 64. Accordingly, each of the grooves 810 followsthe spiral path 869 until blocked by the fluid flow path 64. In this andother aspects, the fluid flow path 64 and the grooves 410 (or, invarious aspects, others of the flow constrictions 310) are separated bya barrier or gap so that fluid in the fluid flow path 64 is restrictedfrom flowing to the grooves 410 by capillary pressure.

Various aspects using grooves 410 around the fluid flow path 64 maintainand control the meniscus shape of the wash fluid 301 so that a higherdome will be formed above the fluid flow path 64 and the wash fluid 301will be restricted from spreading beyond the grooves 410.

Referring to FIGS. 9 and 10, there are shown elevational sections of anexemplary lateral-flow assay device 900 in accordance with at least oneembodiment. FIGS. 9 and 10 illustrate stages in which fluid fills aninternal volume of the lateral-flow assay device 900. The exemplarylateral-flow assay device 900 does not use grooves 410, FIG. 8, as itsflow constriction(s) 310, FIG. 3. Instead, the lateral-flow assay device900 includes a cover 990 arranged over the substrate 9. The cover 990includes the hydrophilic surface 908 facing the substrate 9. Thesubstrate can also have a hydrophilic surface 308, but this is notrequired. The cover 990 also includes an aperture 920 of diameter ddefining a wash port 930 at least partly aligned with the wash additionzone 409. The aperture 920 is configured to receive the wash fluid 301.

At least one of the flow constriction(s) 310 comprises a first coverflow constriction 910, including a protrusion 911 (e.g., a nozzle ornub; examples are discussed below) extending from the cover 990 towardsthe substrate 9 proximate the aperture 920. In the example shown, thefirst cover flow constriction 910, and specifically the protrusion 911,includes a lip of the aperture 920 protruding to a first predetermineddistance h1 from the substrate 9. Also in the example shown, a secondcover flow constriction 912 is arranged outside the aperture 920 andincludes a protrusion 913 extending to a second predetermined distanceh2 from the substrate 9. The second predetermined distance h2 can begreater than the first predetermined distance h1, as shown. In otherconfigurations, h2>h1, h2≈h1, or h2=h1. As used herein, “higher” or“deeper” cover protrusions are those that extend relatively farther fromthe cover; “shorter” or “shallower” cover protrusions are those thatextend relatively less far from the cover 990.

The example of FIGS. 9 and 10 can represent a nozzle (the cover flowconstriction 910 with the interior aperture 920) having an insidediameter d. The nozzle can convey the wash fluid 301 from a fluid supply(not shown; e.g., a pipette or blister) to the hydrophilic surface 908or to the hydrophilic surface 308 (if present). The nozzle can beannular in plan, e.g., a ring structure. The second cover flowconstriction 912 can be an outer ring. Outside the double outer ringstructure (cover flow constrictions 910, 912), the gap distance betweenthe hydrophilic surface 908 and the facing surface of the substrate 9 ish3, which is larger than h2 in this example. The inner ring (theprotrusion 911) advantageously retains the wash fluid 301 in thereservoir 535 when the delivered fluid volume is small, e.g., 5 to 7 μL.The outer ring (the protrusion 913) is spaced farther from the surfaceof the substrate 9 (h2>h1) so that more fluid, e.g., 15 to 17 μL, can beretained within a limited spatial extent (e.g., a diameter of the washaddition zone 409 substantially equal to 5 mm).

Referring specifically to FIG. 9, in an example, d=2 mm, h1=0.35 mm,h2=0.8 mm, and h3=1 mm. The outside diameter of the protrusion 911 is 3mm. The inner diameter of the protrusion 911 is 4 mm, and the outerdiameter of the protrusion 913 is 5 mm. Under the protrusion 911, thevolume of the gap is about 2.5 μL. The gap volume between theprotrusions 911, 913 is 5.5 μL. The gap volume under the protrusion 913is 4.6 μL. The total volume under the three parts is 12.6 μL. Sincemeniscus shape is not exactly straight, experiments showed that thefeature can maintain stability and provide normal wash for a wash volumeof 5 to 20 μL.

FIG. 9 shows the reservoir 535 when a relatively smaller amount of thewash fluid 301 has been added compared to FIG. 10. In this example, thehydrophilic surface 308 is used. The rounded end of the protrusion 911permits the wash fluid 301 to form a dome in the nozzle (the aperture920) and more readily contact the hydrophilic surface 308. Specifically,the rounded end reduces back pressure to permit easier dispensing intothe wash addition area 409 through the aperture 920. A round bottomreduces back pressure by providing an increased radius as the meniscusof the wash fluid 301 moves down the aperture 920 towards the substrate9. This permits the wash fluid 301 to contact the substrate 9 or thehydrophilic surface 308 thereof without a large allied pressure. Thiscan be particularly useful, e.g., with uncoated plastic nozzles withwhich the wash fluid 301 has a contact angle of, e.g., 100°.

Upon contact, the wash fluid 301 wets the hydrophilic surface 308 andthus spreads laterally. The lateral spreading causes the wash fluid 301to also wet the hydrophilic surface 908. Capillary pressure formsmenisci, e.g., a meniscus 935, that define the reservoir 535 as shown.

Referring specifically to FIG. 10, there is shown the reservoir 535 whena relatively larger amount of the wash fluid 301 has been added comparedto FIG. 9, e.g., 15 μL. In this example, the meniscus 1035 is stabilizedby the protrusion 913 (the outer ring). The reservoir 535 is defined bythe meniscus 1035 and a meniscus (shown) inside the aperture 920.

Also in the example of FIG. 10, a first one of the flow constriction(s),e.g., the cover flow constriction 912 including the protrusion 913,includes a proximal edge 1018 and a distal edge 1019 defined withrespect to the fluid flow path 64. The distal edge 1019 is more sharplycurved than the proximal edge 1018. This advantageously increases thedome height at the distal edge 1019, e.g., as discussed above withreference to the angle 531, FIG. 5. In other aspects, the proximal edge1018 is more sharply curved than the distal edge 1019, or the edges1018, 1019 are equally sharply curved. The curvature of the edges 1018,1019 can be selected to determine the volume that can be held thereservoir 535 when the menisci 1035 are retained at the respective oneof the edges 1018, 1019. Increasing the sharpness of curvature of theedges 1018, 1019 increases the effectiveness with which the edges 1018,1019 “pin” (retain) menisci.

FIG. 11A is a sectioned perspective of a lateral-flow assay device 1100according to various aspects, and FIG. 11B is an elevational sectionalong the line XIB-XIB in FIG. 9A. In the section shown in FIG. 11B,dimensions are given in millimeters. As shown, the lip of the aperture920 is substantially annular in shape. Since capillary force naturallytries to maintain circular configurations, using an annular nozzle canadvantageously improves stability of menisci such as that shown in FIG.9. Also as shown, in this example, the aperture 920 and the lip of theaperture (the protrusion 911) are coaxial to one another.

In this example, the cover 990 of the lateral-flow assay device 1100 isarranged over the substrate 9. At least one of the flow constriction(s)310 includes the nozzle 1120 extending from the cover 990 towards thesubstrate 9 and spaced apart from the substrate 9. The nozzle defines awash port 930, FIG. 9, at least partly aligned with the wash additionzone 409, FIG. 9, and configured to receive the wash fluid 301, FIG. 9.At least one said flow constriction 310 can include an annulus (thecover flow constriction 912) arranged around the nozzle 1120 andextending a smaller distance from the cover 990 than does the nozzle1120. The aperture 920 in the nozzle 1120 can be conical, as shown. Thiscan provide humans dispensing the wash fluid 301 through the nozzle 1120a larger target to hit, reducing the probability of spilling the washfluid 301. This can also assist in drawing the wash fluid 301 towardsthe substrate 9, since the reduction in diameter of the aperture 920causes the capillary pressure pulling the wash fluid 301 down near thebottom of the aperture 920 to exceed the capillary pressure pulling thewash fluid 301 up near the wider top of the aperture 920. Alternatively,the nozzle 1120 can have a cylindrical or rectilinear aperture 920, oran aperture 920 of another shape.

In various aspects such as that shown in FIGS. 11A and 11B, nozzle(s)1120 and groove(s) 420 are used together. The nozzles(s) 1120 and thegroove(s) 420 both assist in maintaining meniscus stability andrestricting the metered wash fluid 301 from spreading across thehydrophilic surface 308 of the substrate 9. Various such aspects arediscussed below with reference to FIGS. 12, 13, and 15-19. Moreover,various exemplary configurations of flow constrictions are describedbelow. Unless otherwise specified, flow constrictions shown onsubstrates or on covers can be used independently or can be usedtogether in any combination.

Referring to FIG. 12, there is shown an elevational section of anexemplary lateral-flow assay device 1200 and an illustration of effectsof contact angle. In this example, the lateral-flow assay device 1200includes the substrate 9 having the hydrophilic surface 308 facing thecover 990. The one or more flow constriction(s) 310 include one or morerecessed substrate flow constriction(s), in this example the grooves1210. For example, groove(s) 1210 and nozzle(s) 1120 can be usedtogether when the contact angle of the wash fluid 301 on the hydrophilicsurface 308 is less than 40°. At least one of the substrate flowconstriction(s) can be arranged along a substantially arcuate path 411,FIG. 4, disposed substantially about a portion of the fluid flow path64, e.g., as shown in FIG. 11A.

As the wash fluid 301 is added to the lateral-flow assay device 1200through the aperture 920, it wets the hydrophilic surface 908 of thecover 990 and the facing hydrophilic surface 308 of the substrate 9 andforms menisci. In an example, the wash fluid 301 creeps along thehydrophilic surface 908 on the underside of the cover 990. The shape ofthe meniscus and thus the lateral extent of the reservoir 535 for agiven volume of the wash fluid 301 can be controlled by selectingmaterials having desired contact angles. In an example in which the washfluid 301 has a contact angle of less than 45° with the hydrophilicsurfaces 308, 908, one or more menisci 1235 form. In an example in whichthe wash fluid 301 has a contact angle of greater than 45° with thehydrophilic surfaces 308, 908, one or more menisci 1237 form. As shown,the menisci 1237 extend farther from the aperture 920 than do themenisci 1235. Accordingly, in various aspects, the compositions of thewash fluid 301 and the hydrophilic surfaces 308, 908 are selected toprovide a desired lateral extent of the reservoir 535.

Moreover, the sizes and positions of the substrate flow constriction(s),e.g., the groove(s) 1210, can be selected to cooperate with the nozzle1120. In this example, four grooves 1210, 1211, 1212, 1213 are visible(referred to collectively with reference number 1210). The grooves 1210are configured so that the grooves 1210 farther from the aperture 920will participate in forming reservoirs with larger volumes than thegrooves 1210 closer to the aperture. For example, the groove 1213 canretain a meniscus behind which more of the wash fluid 301 is held thancan the groove 1211. In this non-limiting example, meniscus 1235 is heldby the proximal edge (for clarity, not labeled) of the groove 1212, andthe meniscus 1237 is held by the distal edge of the groove 1213.

In various aspects, the wash fluid 301 can form a stabilized meniscus ata location at which the gap size, i.e., the distance between thehydrophilic surfaces 308, 908, is smaller at that location than atadjacent locations. The grooves 1210 cause this to be the case for theraised areas between the grooves, and the cover flow constrictions causethis to be true between the cover flow constrictions and the hydrophilicsurface 308.

FIG. 13 is an elevational section of an exemplary lateral-flow assaydevice 1300 illustrating stages in which the wash fluid 301 fills aninternal volume of the lateral-flow assay device 1300. For clarity, thestages are indicated with circled numbers, and menisci are indicatedwith dotted curves. Each of stages 2, 3, and 4 includes the wash fluid301 in the areas marked indicated by earlier stages, starting from stage2.

In stage 1, the wash fluid 301 is retained within the nozzle 1120 andforms a dome, as described above.

In stage 2, the wash fluid 301 is retained between the protrusion 911(the lip of the nozzle 1120) and the hydrophilic surface 308. Themenisci are concave. In an example, the reservoir 535 holds about 5 μLin stage 2.

In stage 3, more of the wash fluid 301 has been added. The volume of thereservoir 535 has expanded, so the menisci between the hydrophilicsurface 308 and the protrusion 311 are convex rather than concave. As aresult, the reservoir 535 holds, e.g., about 7 μL in stage 3.

In stage 4, more of the wash fluid 301 has been added, and the reservoir535 has expanded to the menisci 1335. In an example, the reservoir 535holds about 20 μL in stage 4.

FIG. 13 shows one example of a configuration of flow constrictions 310,FIG. 3, that provides a reservoir 535 with a selected capacity in eachof a selected number of steps. The number and arrangement of the flowconstrictions 310, e.g., the nozzle 1120 or other cover flowconstrictions, or the grooves 1210 or other substrate flowconstrictions, can be selected to effectively retain the wash fluid 301in the reservoir 535 above the fluid flow path 64. For example, the flowconstrictions 310 can be configured to effectively retain volumes of thewash fluid 301 in 2 μL increments. Each set of flow constrictions, e.g.,each ring protruding from the hydrophilic surface 908, provides a rangeof stable volumes of the reservoir 535. In this example, the protrusion911 provides stable ones of the reservoirs 535 between volumes of 5(stage 2) and 7 μL (stage 3). These ranges, and configurations usingmultiple flow constrictions, increase the range of possible uses of asingle design of the lateral-flow assay device 1300.

FIG. 14 is an elevational section of another exemplary lateral-flowassay device 1400. The lateral-flow assay device 1400 includes thenozzle 1120 having a lip 1411 (a cover flow constriction 910, FIG. 9).The lip 1411 has a distal surface 1420 with respect to the aperture 920.The distal surface 1420 is sloped and does not have a sharply-curvededge. Capillary pressure will tend to retain the wash fluid 301, FIG.13, in the reservoir 535 as long as the menisci 1435, 1436 contact thesloped distal surface 1420. Since capillary pressure is stronger innarrower apertures, if the reservoir 535 moves, e.g., right, thecapillary pressure pulling the meniscus 1435 to the left will increaseand the capillary pressure pulling the meniscus 1436 to the right willdecrease, returning the reservoir 535 to a more central position.

FIGS. 15-27 are perspectives of components of lateral-flow assay devicesaccording to various aspects. FIGS. 15-18 show examples similar to thosediscussed above with reference to FIGS. 11A-11B. FIG. 15 shows aconfiguration with a single ring (the lip of the nozzle) spaced apartfrom the surface of the substrate 9 by 0.75 mm. FIG. 16 shows aconfiguration similar to that of FIG. 15, but with the ring spaced apartfrom the substrate by 0.35 mm. This configuration can be useful, e.g.,for lateral-flow assay devices designed for only a single volume of thewash fluid 301. FIG. 17 shows a configuration having two rings, eachspaced apart by 0.35 mm. FIG. 18 shows a configuration having two rings,the inner (the lip of the nozzle) spaced apart by 0.75 mm and the outerspaced apart by 0.35 mm. Exemplary devices were constructed according toconfigurations shown in FIGS. 15-18 and were tested. The results aregiven in Table 2, below.

Table 2 shows the wash performance of the four wash feature designsshown in FIGS. 15-18 at different wash volumes. The wash fluid used inthis test was POC wash having properties listed below in Table 3. InTable 2, “overflow” signifies that wash fluid flowed above the fluidflow path 64 (this is undesirable since the wash efficiency will bepoor). “Meniscus out” signifies that the fluid meniscus extendslaterally at least partly beyond the third wash groove 410, FIG. 4, inthe tested lateral-flow assay device. “Off-center” signifies that thefluid meniscus is not centered in the tested arcuate grooves 410. “Good”signifies that the wash fluid 301 is stable in the reservoir 535, FIG.5, and the meniscus is substantially a desired size. Cells in Table 2marked “*” represent preferred embodiments.

TABLE 2 experimental results Gaps Metering volume and fluid types InnerOuter *5 μL *10 μL *15 μL 20 μL 25 μL FIG. ring ring POC POC POC POC POC15 0.75 N/A overflow *good good stable, brief stable, brief overflowoverflow, meniscus out 16 0.35 N/A *stable, fluid *good *good stable,long stable, long short overflow overflow, meniscus out 17 0.35 0.35*stable, fluid *good *good *stable, brief stable, long short overflowoverflow, meniscus out 18 0.75 0.35 stable, off- *stable, brief *stable,brief *stable, brief stable, brief center, brief overflow overflowoverflow overflow, overflow meniscus out

FIG. 17 illustrates a configuration in which at least one of the flowconstriction(s) 310 includes an annulus 1710 arranged around the nozzle1120 and extending substantially the same distance from the cover 990 asdoes the nozzle 1120.

In an example (not shown), the annulus 1710 can be interruptedperiodically, e.g., every 90° around the annulus 1710, thus forming aplurality of independent arcuate protrusions.

FIG. 18 illustrates a configuration in which at least one of the flowconstriction(s) 310 includes an annulus 1810 arranged around the nozzle1120 and extending a larger distance from the cover 990 than does thenozzle 1120.

FIG. 19 is a top perspective view of components of a lateral-flow assaydevice according to various aspects. In the illustrated configuration,the aperture 1920 of the nozzle 1120 and the lip 1911 of the aperture1920 are axially offset from one another. The wash fluid 301 is shownfilling the aperture 1920 and being dispensed onto the substrate 9. Forclarity of explanation, the axes of the lip 1911 and of the aperture1920 are shown, as is the offset 1995 between them in this example.Axial offset provides increased flexibility in the design of thelateral-flow assay device 1900, since the location at which the washfluid 301 is received (the aperture 1920) can be offset from thelocation at which the wash fluid 301 is dispensed onto the substrate 9.In an aspect, the lowest tip of the aperture 920 is disposed above thefluid flow path 64 to be washed. Also as shown, the aperture 1920 canhave a partly-conical, partly-cylindrical shape.

FIG. 20 is a perspective of components of the lateral-flow assay device1900, FIG. 19, according to various aspects. FIG. 20 shows a bottomperspective view of the nozzle 1120. As shown, the nozzle 1120 has aconical portion, as indicated. FIG. 20 also shows a portion of thehydrophilic surface 908. Accordingly, in various embodiments, a firstone of the flow constriction(s) 310 is shaped substantially as a convexclosed figure such as a cone. Convex closed figures can include nubs,e.g., circular, elliptical, or polygonal in planwise cross-section.

FIG. 21 is a bottom perspective view of components of a lateral-flowassay device according to various aspects. In the illustratedconfiguration, at least one of the flow constrictions 310 includes aprotrusion 2112 spaced apart from the nozzle 1120. The protrusion 2112permits menisci to form to differentially attract to a known locationany excess wash fluid beyond the amount that can be held in a reservoir535, FIG. 5, formed by the nozzle 1120 alone. This can advantageouslypermit, e.g., drawing excess amounts of the wash fluid 301 away from thefluid flow path 64 or a portion thereof.

FIG. 22 is a bottom perspective view of components of a lateral-flowassay device according to various aspects. In the illustratedconfiguration, a nozzle 2220 has a stepped surface 2225 facing thesubstrate 9, FIG. 19. This advantageously provides defined locations atwhich menisci will preferentially form, e.g., the edges of the steps.

FIG. 23 is a bottom perspective view of components of a lateral-flowassay device according to various aspects. In the illustratedconfiguration, at least one of the flow constrictions 310 is aprotrusion 2312 is spaced apart from the nozzle 2220. The protrusion2312 can attract excess volumes of the wash fluid 301, e.g., asdescribed above with reference to FIG. 21.

FIGS. 24-27 are bottom perspective views of respective covers 990 ofvarious exemplary lateral-flow assay devices. Each of the covers 990includes the respective hydrophilic surface 908.

FIG. 24 is a bottom perspective view of components of a lateral-flowassay device according to various aspects. In the illustratedconfiguration, the nozzle 2420 has a relatively broad plateau 2425surrounding a relatively narrow aperture 920. The aperture 920 can bebroader where the wash fluid 301 is added to the aperture 920, e.g., asshown in FIG. 19. The plateau 2425 is one of the flow constriction(s)310 in this example. The plateau 2425 can be, e.g., 1.78 mm in diameter.

FIG. 25 is a bottom perspective view of components of a lateral-flowassay device according to various aspects. In the illustratedconfiguration, similar to the configuration shown in FIG. 18, at leastone of the flow constriction(s) 320 includes an annulus 2520 arrangedaround the nozzle 2420 and extending a larger distance from the cover990 than does the nozzle 2420. In this example, the outside diameter ofthe annulus 2520 is 3 mm. The annulus 2520 can be concentric with thenozzle 2420, or can be axially offset therefrom. The relative positionsof the annulus 2520 and the nozzle 2420 can be selected to providedesired shapes of the menisci that form when the wash fluid 301 is addedto the lateral-flow assay device.

FIG. 26 is a bottom perspective view of components of a lateral-flowassay device according to various aspects. In the illustratedconfiguration, at least one of the flow constriction(s) 310 includes aplurality of protrusions 2630 arranged substantially symmetrically aboutthe annulus 2520 and spaced apart from the annulus 2520. In thisexample, four of the protrusions 2630 are present, spaced at 90°intervals around the annulus 2520. The annulus 2520, the nozzle 2420,and the protrusions 2630 can have any desired relationship of relativeheight off the cover 990. As described above with reference to FIG. 21,the protrusions 2630 provide increased control of where excess volumesof the wash fluid 301 are stored. In various aspects, the protrusions2630 can all have the same shape or can have any number of differentshapes; any number of the protrusions 2630 can be used; and theprotrusions 2630 can be spaced at any angles, evenly or unevenly.

FIG. 27 is a bottom perspective view of components of a lateral-flowassay device according to various aspects. In the illustratedconfiguration, at least one of the flow constriction(s) 310 includes aplurality of protrusions 2730, 2731 arranged substantially symmetricallyabout the nozzle 2420 and spaced apart from the nozzle 2420. In thisexample, four of the protrusions 2730 are arranged alternating with fourof the protrusions 2731 around the nozzle 2420 at 45° intervals. Solidand dotted lead lines are used for clarity only and without limitation.In various aspects, the protrusions 2730, 2731 can all have the sameshape or can have any number of different shapes; any number of theprotrusions 2730, 2731 can be used; and the protrusions 2730, 2731 canbe spaced at any angles, evenly or unevenly.

The configurations shown in FIGS. 26 and 27 can be useful forlateral-flow assay devices using high volumes of the wash fluid 301compared to, e.g., the configurations shown in FIGS. 24 and 25.

FIGS. 28-36 are graphical representations of photographs of stages inexperimental tests of an exemplary lateral-flow assay device accordingto various aspects. The tested exemplary lateral-flow assay device wasconfigured as shown in FIGS. 11A-11B,

Experiment 1 (FIGS. 28-30), experiment 2 (FIGS. 31-33), and experiment 3(FIGS. 34-36) illustrate that various flow constriction(s) 310 can,together with the hydrophilic surfaces 308, 908, effectively deliverdifferent wash fluids (water, POC wash and NDSB Wash) to accomplish washeffectively and maintain the stability of wash fluid menisci within thewash addition area 409 in the lateral-flow assay device during an assayfluid flow process. The properties of the tested wash fluids 301 arelisted in Table 3:

TABLE 3 Wash Fluid Viscosity (cP) Surface Tension (dynes/cm) DI Water0.88 72.7 POC Wash 1.0 0.93 32.8 NDSB (FlumAb) 0.92 31.9

Referring to FIGS. 28-30, there are shown stages in Experiment 1. Thesample 101, FIG. 1, was 1% silwet surfactant in plasma and included redfood dye for visibility. Eight microliters of the sample 101 were addedto the sample addition zone 2, FIG. 1. Once the sample 101 filled about40% of the volume of the wicking zone 5, FIG. 1, 17 □L of POC wash fluid(at room temperature) with blue food dye was added to the wash additionzone 409. Red food dye is added to the sample, and blue dye is added tothe wash fluid. FIGS. 28-30 show fluid flow and wash patterns at threedifferent stages of the tested assay process with wash addition.

FIG. 28 shows the sample 101 (red color) having filled about 40% of thevolume of the wicking zone 5 prior to wash addition. FIG. 28 shows thetested lateral-flow assay device immediately after adding the wash fluid301 (blue color). The grooves 410, FIG. 4, are retaining the wash fluid301. FIG. 30 shows the distribution of the wash fluid 301 distributionwhen fluid, in this test the sample 101, reaches the end 3005 of thewicking zone 5. In this experiment, the fluid of the sample 101 wascompletely displaced by the wash fluid in the detection zone channel3064 of the fluid flow path 64, FIG. 3. Moreover, the wash fluidextended into the wicking zone 5 in a region 3001. The fluid under thewash addition zone 409 is still pinned within the grooves 410 and isstill stable after the wash flow is complete.

Referring to FIGS. 31-33, there are shown stages in Experiment 2. Thesample 101 as in Experiment 1 was added to the sample addition zone 2.FIG. 31 shows the sample 101 (red color) having filled about 30% of thevolume of the wicking zone 5 prior to wash addition. At that point, thewash fluid 301 was added. FIG. 32 shows the tested lateral-flow assaydevice immediately after adding the wash fluid 301, in this experiment17 □L, de-ionized water (at room temperature) plus green food dye (greencolor). The wash fluid 301 (green color) is retained within the grooves410. FIG. 33 shows the lateral-flow assay device when fluid, in thisinstance the sample 101, reached the end 3005 of the wicking zone 5. Thefluid of the sample 101 (red color) is completely displaced by the washfluid 301 (green color) in the detection zone channel 3064. Moreover,the wash fluid extended into the wicking zone 5 in a region 3301. Thewash fluid 301 in the wash addition zone 409 is still pinned within thegrooves 410 and is still stable after the wash flow is complete.

Referring to FIGS. 34-36, there are shown stages in Experiment 3. Thesample 101 as in Experiment 1 was added to the sample addition zone 2.FIG. 34 shows the sample 101 (red color) having filled about 60% of thevolume of the wicking zone 5 prior to wash addition. At that point, thewash fluid 301 was added. FIG. 35 shows the tested lateral-flow assaydevice immediately after adding the wash fluid 301, in this experiment17 □L NDSB Wash fluid (at room temperature) plus blue food dye (bluecolor). The wash fluid 301 (blue color) is retained within the grooves410, FIG. 4. FIG. 36 shows the lateral-flow assay device when fluid, inthis instance the sample 101, reached the end 3005 of the wicking zone5. The fluid of the sample 101 (red color) is completely displaced bythe wash fluid 301 (blue color) in the detection zone channel 3064.Moreover, the wash fluid extended into the wicking zone 5 in a region3601. The wash fluid 301 in the wash addition zone 409 is still pinnedwithin the grooves 410 and is still stable after the wash flow iscomplete.

Various experiments were conducted for the configurations shown in FIGS.24-27 using POC wash fluid. For all four of those tested configurations,wash was performed effectively for all three tested dispense volumes(10, 15 and 20 μL) of the POC wash fluid 301. The wash fluid 301 wasclearly visible in the detection zone channel 3064 and the wicking zone5. In some configurations, the wash fluid 301 moved only downstream ifthe sample 101 was not touching the cover 990. In some configurations,the wash fluid moved both upstream and downstream if the sample 101touched the cover 990. All tested configurations provided stable meniscifor volumes of the wash fluid 301 of 10 μL and 15 μL. The wash fluid 301was retained within the third (outermost) ring of the grooves 410 atthose volumes. For a volume of 20 μL, the wash fluid 301 passed thethird ring in some tests. In one test, non-stable meniscus behavior wasobserved. Accordingly, the flow constrictions 310 can be designed basedon the volumes of the sample 101 and the wash fluid 301 to providestable meniscus behavior. In various tested configurations using nubs(e.g., the protrusions 2630, FIG. 26), the nubs did attract thedispensed wash fluid 301. The menisci were not symmetric in every test.Accordingly, the flow constrictions 310 can be designed based on thevolumes of the wash fluid 301 and the configuration of the fluid flowpath 64 to provide menisci with a desired degree of symmetry.

Referring to FIG. 37, there is shown an apparatus 3700 for analyzing afluidic sample 101 according to at least one exemplary embodiment. Theapparatus 3700 includes a transport system 3710 for conveying thelateral-flow assay device 300 between components described below. Forsimplicity, the transport system 3710 is represented as a continuousconveyor belt. However, this is not limiting. The transport system 3710can include conveyor(s), gripper(s), robotic arm(s), or other device(s)for moving the lateral-flow assay device 300 with respect tobelow-described components, or can include stage(s), conveyor(s), orother device(s) for moving below-described components with respect tothe lateral-flow assay device 300, in any combination. Various examplesof the transport system 3710 are described in commonly-assigned U.S.Pat. No. 8,080,204 to Ryan et al. and U.S. Pat. No. 8,043,562 to Tomassoet al., each of which is incorporated herein by reference, and in U.S.Pat. No. 7,632,468 to Barski, et al, incorporated herein by reference.Positions of the lateral-flow assay device 300 at various stages ofprocessing are shown in phantom.

In this example, the lateral-flow assay device 300 includes the sampleaddition zone 2, the wash addition zone 409, and the wicking zone 60disposed in that order along the fluid flow path 64, e.g., as discussedabove with reference to FIG. 3. Any of the above-described embodimentsof lateral-flow assay devices can be used in addition to or in place ofthe lateral-flow assay device 300, e.g., the lateral-flow assay devices300, 400, 700, 800, 900, 1100, 1200, 1300, 1400, 1900, or otherillustrated or described lateral-flow assay devices.

A sample-metering mechanism 3720 is configured to selectively apply thefluidic sample 101 to the sample addition zone 2 of the at least onelateral-flow assay device 300. The illustrated sample-metering mechanism3720 includes a disposable metering tip 3724 holding, e.g., 250 μL ofthe fluidic sample 101. In various aspects, there is a one-to-onecorrespondence between a particular fluidic sample 101 and a particulardisposable metering tip 3724. In an example, each metering event metersbetween ˜5 μL and ˜10 μL of the fluidic sample 101.

In the illustrated example, and for explanation only, thesample-metering mechanism 3720 includes a piston 3721 and a drivingsystem 3722 operating the piston 3721 to dispense a selected volume ofthe fluidic sample 101 from the metering tip 3724. Other structures formetering can also be used, e.g., air or fluid pressure sources orpiezoelectric or thermal actuators. An exemplary metering tip 3724 isdescribed in U.S. Publication No. 2004/0072367 by Ding, et al., thedisclosure of which is incorporated herein by reference. Metering thesample 101 onto a lateral-flow assay device 100 is referred to herein as“spotting.”

The exemplary apparatus 3700 further includes the wash-meteringmechanism 3725 configured to selectively apply the wash fluid 301 to thewash addition zone 409 of the lateral-flow assay device 300. In anexample, the wash-metering mechanism 3725 includes a metering nozzle3726 and an actuator (not shown), e.g., a piston such as the piston3721. In another example, the wash-metering mechanism includes ablister.

The wash addition zone 409 includes one or more flow constriction(s) 310spaced apart from the fluid flow path 64 to form a meniscus in theapplied wash fluid. Examples of the wash addition zones 409 and the flowconstrictions 310 are discussed above with reference to FIGS. 3-27. Asdiscussed above, the fluid flow path 64 is configured to draw theapplied wash fluid 301 out of a reservoir 535, FIG. 5, defined at leastpartly by the meniscus.

The exemplary apparatus 3700 includes at least one incubator 3730.Various types of sample testing, including potentiometric, ratechemistry, and endpoint tests, may be required for any given patientsample, necessitating both different incubation intervals and differenttest apparatus within the incubator 3730. Accordingly, more than oneincubator, or a tandem or other multi-test-capable incubator can beused. For clarity, only one incubator 3730 is shown. Various examples ofthe incubators 3730 and related components are described in U.S. Pat.Nos. 4,287,155 and 7,312,084 to Jakubowicz, et al., entitled “TandemIncubator for Clinical Analyzer,” each of which is hereby incorporatedby reference in its entirety.

The incubator 3730 retains the lateral-flow assay device(s) 300, e.g.,at room temperature or under selected environmental conditions, until anaccurate measurement can be taken. Some lateral-flow assay devices 300require endpoint testing, which requires only a single read be performedfollowing a predetermined incubation interval (e.g., approximately 5minutes). Other lateral-flow assay devices 300, such as those requiringrate chemistries, require a number of reads to be taken throughout thecourse of incubation. The incubator 3730 or the transport system 3710can therefore include structures for transporting lateral-flow assaydevice(s) 300 between the incubator 3730 and a measurement device 3740,discussed below.

The exemplary apparatus 3700 shown further includes at least onemeasurement device 3740. The measurement device 3740 can include apotentiometric sensor, e.g., a voltmeter, ammeter, or charge meter, or acolorimetric or other photometric sensor. Exemplary photometric sensorsinclude photodiodes and line-scan or area-scan reflectometers orimagers, e.g., charge-coupled device (CCD) or complementarymetal-oxide-semiconductor (CMOS) imagers. Colorimetric sensors canoperate in reflective or transmissive modes. Reflective colorimetricsensors can be arranged to measure the front or back of the lateral-flowassay device 300.

In an example, the measurement device 3740 includes a light source 3742(represented graphically as a lamp). The light source 3742 can include alamp, light-emitting diode (LED), laser, or other source of opticalradiation. The exemplary measurement device 3740 also includes aphotosensor 3744 that captures light of the light source 3742 reflectedfrom the detection zone 56 of the lateral-flow assay device 300.

The exemplary apparatus 3700 further includes a controller 3786configured to operate each of the sample-metering mechanism 3720, thewash-metering mechanism 3725, and the at least one measurement device3740 in accordance with a predetermined timing protocol in order todetermine at least one characteristic of the applied fluidic sample 101.The controller 3786 is configured to operate the wash-metering mechanism3725 after operating the sample-metering mechanism 3720. The controller3786 can also be configured to operate the incubator 3730.

For clarity only, communications connections between the controller 3786and other components are shown dashed. Further and according to thisexemplary embodiment, the controller 3786 is configured to operate thetransport system 3710. For example, the controller 3786 can sequence themotion of the lateral-flow assay device 300 through the sample-meteringmechanism 3720, the incubator 3730, and the at least one measurementdevice 3740 to perform a potentiometric or colorimetric measurement ofthe fluidic sample 101. The exemplary controller 3786 can be furtherconfigured to receive data from the photosensor 3744 and provide agraphical representation of the measured data via an electronic display.The controller 3786 can include various components discussed below withreference to FIG. 39, e.g., a processor 3986.

FIG. 38 shows a flowchart illustrating an exemplary method fordisplacing a fluidic sample in a fluid flow path of an assay device. Inat least one example, processing begins with step 3810. For clarity ofexplanation, reference is herein made to various components shown inFIGS. 1-27, 37 that can carry out or participate in the steps of theexemplary method. It should be noted, however, that other components canbe used; that is, exemplary method(s) shown in FIG. 38 are not limitedto being carried out by the identified components. The method caninclude automatically carrying out the listed steps using a processor,e.g., the processor 3986, FIG. 39, or another processor in thecontroller 3786, FIG. 37.

In step 3810, the fluidic sample 101 is dispensed from a sample supply,e.g., the sample-metering mechanism 3720, FIG. 37, onto a sampleaddition zone 2 of the lateral-flow assay device 300. The dispensedfluidic sample 101 travels along the fluid flow path 64 of thelateral-flow assay device 300.

In step 3820, a wash fluid 301 is dispensed from a wash-fluid supply,e.g., the wash-metering mechanism 3725, FIG. 37, onto a wash additionzone 409 of the lateral-flow assay device 300 downstream of the sampleaddition zone 2 along the fluid flow path 64. A meniscus is then formedin the dispensed wash fluid 301 by at least one flow constriction 310 ofthe lateral-flow assay device 300. The fluid flow path 64 draws at leastsome of the dispensed wash fluid 301 out of the reservoir 535 defined atleast partly by the meniscus. The drawn at least some of the dispensedwash fluid 301 displaces at least some of the fluidic sample 101 in thefluid flow path 64. This is discussed above with reference to FIG. 6.Step 3820 permits performing assays that require washing with other thanfluid of the sample 101 in order to provide accurate results. In variousembodiments, step 3820 is followed by step 3830.

In step 3830, after said dispensing the wash fluid in step 3820, thepresence of a detectable signal corresponding to a characteristic of thedispensed fluid sample 101 is determined. This can be done using theincubator 3730, the measurement device 3740, or both. In embodimentsusing incubation, the incubation time can be selected as appropriatebased on the fluidics and dimensions of the lateral-flow assay device300 and the viscosities or surface tensions of the sample 101 or thewash fluid 301.

FIG. 39 is a high-level diagram showing the components of an exemplarydata-processing system 3901 for analyzing data, operating an apparatus3700, FIG. 37, for analyzing samples 101 and performing other analysesdescribed herein, and related components. The data-processing system3901 includes a processor 3986, a peripheral system 3920, a userinterface system 3930, and a data storage system 3940. The peripheralsystem 3920, the user interface system 3930 and the data storage system3940 are communicatively connected to the processor 3986. The processor3986 can be communicatively connected to a network (not shown). Thefollowing devices can each include one or more of the systems 3986,3920, 3930, 3940, and can each connect to one or more network(s): thecontroller 3786, the sample-metering mechanism 3720, the wash-meteringmechanism 3725, the incubator 3730, the light source 3742, and thephotosensor 3744, all FIG. 37. The processor 3986, and other processingdevices described herein, can each include one or more microprocessors,microcontrollers, field-programmable gate arrays (FPGAs),application-specific integrated circuits (ASICs), programmable logicdevices (PLDs), programmable logic arrays (PLAs), programmable arraylogic devices (PALs), or digital signal processors (DSPs).

The processor 3986 can implement processes of various aspects describedherein. The processor 3986 and related components can, e.g., carry outprocesses for performing assays or for displacing a fluidic sample 101in a fluid flow path 64 of a lateral-flow assay device 300. Examples ofsuch processes are described above with reference to FIGS. 37 and 38.

The processor 3986 can be embodied in one or more device(s) forautomatically operating on data, e.g., a central processing unit (CPU),microcontroller (MCU), desktop computer, laptop computer, mainframecomputer, personal digital assistant, digital camera, cellular phone,smartphone, or any other device for processing data, managing data, orhandling data, whether implemented with electrical, magnetic, optical,biological components, or otherwise.

The phrase “communicatively connected” includes any type of connection,wired or wireless, for communicating data between devices or processors.These devices or processors can be located in physical proximity or not.For example, subsystems such as the peripheral system 3920, the userinterface system 3930, and the data storage system 3940 are shownseparately from the processor 3986 but can be stored completely orpartially within the processor 3986.

The peripheral system 3920 can include one or more devices configured toprovide digital content records to the processor 3986. For example, theperipheral system 3920 can include or communicate with one or moremeasurement device(s) 3740, FIG. 37. The processor 3986, upon receipt ofdigital content records from a device in the peripheral system 3920, canstore such digital content records in the data storage system 3940. Invarious examples, the peripheral system 3920 is communicativelyconnected to one or more of the sample-metering mechanism 3720, thewash-metering mechanism 3725, the incubator 3730, the light source 3742,and the photosensor 3744, all FIG. 37.

The user interface system 3930 can convey information in eitherdirection, or in both directions, between a user 3938 and the processor3986 or other components of the data-processing system 3901. The userinterface system 3930 can include a mouse, a keyboard, another computer(connected, e.g., via a network or a null-modem cable), or any device orcombination of devices from which data is input to the processor 3986.The user interface system 3930 also can include a display device, e.g.,an electronic display 3935, a processor-accessible memory, or any deviceor combination of devices to which data is output by the processor 3986.The user interface system 3930 and the data storage system 3940 canshare a processor-accessible memory.

The data storage system 3940 can include or be communicatively connectedwith one or more processor-accessible memories configured to storeinformation. The memories can be, e.g., within a chassis or as parts ofa distributed system. The phrase “processor-accessible memory” isintended to include any data storage device to or from which theprocessor 3986 can transfer data (using appropriate components of theperipheral system 3920), whether volatile or nonvolatile; removable orfixed; electronic, magnetic, optical, chemical, mechanical, orotherwise. Exemplary processor-accessible memories include but are notlimited to: registers, floppy disks, hard disks, tapes, bar codes,Compact Discs, DVDs, read-only memories (ROM), erasable programmableread-only memories (EPROM, EEPROM, or Flash), and random-access memories(RAMs). One of the processor-accessible memories in the data storagesystem 3940 can be a tangible non-transitory computer-readable storagemedium, i.e., a non-transitory device or article of manufacture thatparticipates in storing instructions that can be provided to theprocessor 3986 for execution.

In an example, the data storage system 3940 includes a code memory 3941,e.g., a RAM, and a disk 3943, e.g., a tangible computer-readable storagedevice such as a hard drive or Flash drive. Computer programinstructions are read into the code memory 3941 from the disk 3943. Theprocessor 3986 then executes one or more sequences of the computerprogram instructions loaded into the code memory 3941, as a resultperforming process steps described herein. In this way, the processor3986 carries out a computer implemented process. For example, steps ofmethods described herein, blocks of the flowchart illustrations or blockdiagrams herein (e.g., FIG. 38), and combinations of those, can beimplemented by computer program instructions. The code memory 3941 canalso store data, or can store only code.

Various aspects described herein may be embodied as systems or methods.Accordingly, various aspects herein may take the form of an entirelyhardware aspect, an entirely software aspect (including firmware,resident software, micro-code, etc.), or an aspect combining softwareand hardware aspects These aspects can all generally be referred toherein as a “service,” “circuit,” “circuitry,” “module,” or “system.”

Furthermore, various aspects herein may be embodied as computer programproducts including computer readable program code stored on a tangiblenon-transitory computer readable medium. Such a medium can bemanufactured as is conventional for such articles, e.g., by pressing aCD-ROM. The program code includes computer program instructions that canbe loaded into the processor 3986 (and possibly also other processors),to cause functions, acts, or operational steps of various aspects hereinto be performed by the processor 3986 (or other processor). Computerprogram code for carrying out operations for various aspects describedherein may be written in any combination of one or more programminglanguage(s), and can be loaded from the disk 3943 into the code memory3941 for execution.

Various above-described embodiments advantageously use flowconstriction(s) 310, FIG. 3, in the wash addition zone 409 to stabilizethe dispensed wash fluid 301, e.g., to pin the wash fluid 301 toselected locations in the wash addition zone 409. The flowconstriction(s) 310 advantageously encourage the formation of one ormore partly-meniscus-delimited reservoir(s) 535 that can receivevariable volumes of the wash fluid 301 with reduced sensitivity to thedispensing rate of the wash fluid 301. Moreover, the pressure of suchmenisci is close to the ambient, reducing the probability of overflowingthe fluid flow path 64.

Various exemplary flow constriction(s) include nozzle(s) that connects awash fluid supply to the fluid flow path 64 in the lateral-flow assaydevice in the wash addition zone 409; very low nozzle outlets to promotecontact between the wash fluid 301 in the nozzle and the hydrophilicsurface 308 on the substrate 9; and steps outside the nozzle (e.g., asin FIG. 22) to permit variable fluid meniscus sizes (volumes of thereservoir 535) while maintaining meniscus stability.

Various aspects advantageously permit variable-rate, variable-amountdelivery of the wash fluid 301, and stabilize the received wash fluid301 at a desired location. Various aspects reduce the probability ofoverflowing the fluid flow path 64, which improves wash efficiency.Various aspects advantageously provide robust wash performance withrespect to one or more of the following properties:

-   -   Variation in the volume of the wash fluid 301 delivered to the        wash addition zone 409 within the range, e.g., from 7 μL to 17        μL. This relaxed volume range can reduce the development cost of        wash fluid delivery system (e.g., the blister).    -   Variation in the delivery rate of the wash fluid 301 within the        range, e.g., 1 μL/sec to >10 μL/sec. This relaxed range also        facilitates more effective fluid delivery system design (e.g., a        burst of wash fluid from a squeezed blister can be used).    -   Maintenance of a stable meniscus in the wash addition zone,        independent of above-noted variations in the delivery volume and        delivery rate of the wash fluid 301.    -   Entry of the wash fluid 301 into the fluid flow path 64 at an        appropriate location to effectively displace the fluid of the        sample 101 in the fluid flow path 64 without “overflow,” i.e.,        the wash fluid 301 flowing over the sample 101 between the        microposts 7 inside the fluid flow path 64.    -   Termination of the fluid flow of the sample 101 when the wash        fluid 301 is added. Various aspects restrict the sample 101 from        flowing along the fluid flow path 64 downstream past the wash        addition zone 409 once the wash fluid 301 is added.    -   Maintenance of meniscus stability in the wash addition zone 409        as the wash fluid 301 enters the fluid flow path 64 to perform        the wash.    -   Variation in the amount of the wash fluid 301 to be delivered        through the detection zone channel 3064 in the range from 1 μL        to 4 □L, or in the range of >4 μL.

PARTS LIST FOR FIGS. 1-39

-   1 lateral-flow assay device-   2 sample addition zone-   3 reagent zone-   4 detection zone-   5 wicking zone-   7 microposts-   9 substrate-   20 lateral-flow assay device-   40 substrate-   44 top surface-   48 sample addition zone-   52 reagent zone-   55 detection channel-   56 detection zone-   57 flow promoter-   60 wicking zone-   64 fluid flow path-   70 hydrophilic layer-   72 vents-   100 lateral-flow assay device-   101 sample-   300 lateral-flow assay device-   301 wash fluid-   308 hydrophilic surface-   310 flow constriction-   311 protrusion-   400 lateral-flow assay device-   409 wash addition zone-   410 groove-   411 arcuate path-   464 centerline-   511 corner-   520 meniscus-   521 angle-   530 meniscus-   531 angle-   535 reservoir-   655 area-   700 lateral-flow assay device-   710 reference point-   764 centerline-   800 lateral-flow assay device-   810 grooves-   869 spiral path-   900 lateral-flow assay device-   908 hydrophilic surface-   910 cover flow constriction-   911 protrusion-   912 cover flow constriction-   913 protrusion-   920 aperture-   930 wash port-   935 meniscus-   990 cover-   1018 proximal edge-   1019 distal edge-   1035 meniscus-   1100 lateral-flow assay device-   1120 nozzle-   1200 lateral-flow assay device-   1210, 1211, 1212, 1213 grooves-   1235, 1237 menisci-   1300 lateral-flow assay device-   1335 meniscus-   1400 lateral-flow assay device-   1411 lip-   1420 distal surface-   1435, 1436 menisci-   1710, 1810 annuli-   1900 lateral-flow assay device-   1911 lip-   1920 aperture-   1995 offset-   2112 protrusion-   2220 nozzle-   2225 stepped surface-   2312 protrusion-   2420 nozzle-   2425 plateau-   2520 annulus-   2630, 2730, 2731 protrusions-   3001 region-   3005 end-   3064 detection zone channel-   3301, 3601 regions-   3700 apparatus-   3710 transport system-   3720 sample-metering mechanism-   3721 piston-   3722 driving system-   3724 disposable metering tip-   3725 wash-metering mechanism-   3726 metering nozzle-   3730 incubator-   3740 measurement device-   3742 light source-   3744 photosensor-   3786 controller-   3810, 3820, 3830 steps-   3901 data-processing system-   3920 peripheral system-   3930 user interface system-   3935 electronic display-   3938 user-   3940 data storage system-   3941 code memory-   3943 disk-   3986 processor-   F flow direction

The invention is inclusive of combinations of the aspects describedherein. References to “a particular embodiment” (or “aspect” or“version”) and the like refer to features that are present in at leastone aspect of the invention. Separate references to “an embodiment” or“particular embodiments” or the like do not necessarily refer to thesame embodiment or embodiments; however, such embodiments are notmutually exclusive, unless so indicated or as are readily apparent toone of skill in the art. The use of singular or plural in referring to“method” or “methods” and the like is not limiting. The word “or” isused in this disclosure in a non-exclusive sense, unless otherwiseexplicitly noted. The invention has been described in detail withparticular reference to certain preferred aspects thereof, but it willbe readily apparent that other modifications and variations are possiblewithin the intended ambits of the concepts described herein and inaccordance with the following claims.

The invention claimed is:
 1. A lateral-flow assay device comprising: a)a substrate having a top surface including a sample addition zone and awash addition zone disposed along a fluid flow path through which asample flows under capillary action in a downstream direction away fromthe sample addition zone and towards the wash addition zone, wherein thefluid flow path is defined by a plurality of projections extending fromthe top surface of the substrate, the plurality of projections having aheight, diameter and center to center spacing that creates lateralcapillary force to an applied liquid and in which a wash fluid can beadded to the device in the wash addition zone; b) at least onehydrophilic surface arranged in the wash addition zone; and c) flowconstriction(s) including a plurality of spaced grooves formed in thetop surface of the substrate that are spaced apart from and form anarcuate path around the fluid flow path of the wash addition zone andarranged to define, with the at least one hydrophilic surface, areservoir configured to retain the wash fluid by formation of a meniscusbetween the at least one hydrophilic surface and the one or more flowconstriction(s) in which an outer edge of the meniscus latches to anedge of one of the grooves; and in which the plurality of theprojections of the fluid flow path draw wash fluid from the definedreservoir, which is in fluid contact with the projections.
 2. Thelateral-flow assay device as recited in claim 1, wherein the grooves aredisposed about a reference point along a centerline of the fluid flowpath extending through the wash addition zone.
 3. The lateral-flow assaydevice as recited in claim 1, including a plurality of the flowconstriction(s), each of the flow constrictions including groove(s)formed in the hydrophilic surface, said groove(s) being arranged alongrespective arcuate paths about the centerline of the fluid flow path. 4.The lateral-flow assay device as recited in claim 1, wherein at leastone said groove has a substantially rectangular cross-section.
 5. Thelateral-flow assay device as recited in claim 1, said flowconstriction(s) including at least three spaced-apart grooves formed inthe top surface of the substrate.
 6. The lateral-flow assay device asrecited in claim 1, wherein said at least one groove is configured as asegment of a spiral.
 7. The lateral-flow assay device as recited inclaim 1, further including a cover arranged over the substrate, thecover including the at least one hydrophilic surface facing thesubstrate and an aperture defining a wash port at least partly alignedwith the wash addition zone that receives the wash fluid, in which theflow constrictions further comprise a first cover flow constrictionincluding a protrusion extending from the cover towards the substrateproximate the aperture.
 8. The lateral-flow assay device as recited inclaim 7, wherein the first cover flow constriction includes a lip of theaperture protruding to a first predetermined distance from thesubstrate.
 9. The lateral-flow assay device as recited in claim 8,wherein a second cover flow constriction includes a protrusion extendingto a second predetermined distance from the substrate.
 10. Thelateral-flow assay device as recited in claim 9, wherein the secondpredetermined distance is greater than the first predetermined distance.11. The lateral-flow assay device as recited in claim 9, wherein thesecond cover flow constriction is arranged outside the aperture.
 12. Thelateral-flow assay device as recited in claim 8, wherein the lip of theaperture is substantially annular in shape.
 13. The lateral-flow assaydevice as recited in claim 8, wherein the aperture and the lip of theaperture are coaxial to one another.
 14. The lateral-flow assay deviceas recited in claim 8, wherein the aperture and the lip of the apertureare axially offset from one another.
 15. The lateral-flow assay deviceas recited in claim 7, wherein the first cover flow constriction isshaped as a convex closed figure.
 16. The lateral-flow assay device asrecited in claim 7, wherein the first cover flow constriction includes aproximal edge and a distal edge defined with respect to the fluid flowpath wherein the distal edge is defined by a curve which is sharper thanthe curve defining the proximal edge.
 17. The lateral-flow assay deviceas recited in claim 1, further including a cover arranged over thesubstrate having at least one cover flow constriction and in which theat least one cover flow constriction comprises a nozzle extending fromthe cover towards the substrate, defining a wash port at least partlyaligned with the wash addition zone, for receiving the wash fluid. 18.The lateral-flow assay device as recited in claim 17, at least one saidflow constriction comprising an annulus arranged around the nozzle andextending a smaller distance from the cover than does the nozzle. 19.The lateral-flow assay device as recited in claim 17, at least one saidflow constriction comprising an annulus arranged around the nozzle andextending substantially the same distance from the cover as does thenozzle.
 20. The lateral-flow assay device as recited in claim 17,wherein the at least one said cover flow constriction comprises anannulus arranged around the nozzle and extending a larger distance fromthe cover than does the nozzle.
 21. The lateral-flow assay device asrecited in claim 20, wherein the at least one said cover flowconstriction further comprises a plurality of protrusions arrangedsymmetrically about the annulus and spaced apart from the annulus. 22.The lateral-flow assay device as recited in claim 17, wherein the nozzlehas a surface defined by a plurality of annular steps facing thesubstrate.
 23. The lateral-flow assay device as recited in claim 17, inwhich the at least one said cover flow constriction comprises aprotrusion spaced apart from the nozzle.
 24. The lateral-flow assaydevice as recited in claim 17, wherein the at least one said cover flowconstriction comprises a plurality of protrusions arranged substantiallyabout the nozzle and spaced apart from the nozzle.
 25. The lateral-flowassay device as recited in claim 1, further including at least onedetection zone disposed along the fluid flow path downstream of thesample addition zone and the wash addition zone, the at least onedetection zone including a detection material responsive to an analyteof the sample to produce a detectable signal.